JP4897814B2 - Poloxamer quantification method - Google Patents

Abstract

The invention relates to analytical determination of poloxamers in a liquid sample of protein.

Description

  The present invention relates to the field of analytical measurement of surfactants belonging to poloxamers in liquid protein samples.

  Poloxamers are nonionic block copolymers of ethylene oxide (EO) and propylene oxide (PO). These are used in pharmaceutical formulations as surfactants, emulsifiers, stabilizers, and dispersants.

  A well-known analytical method for characterizing poloxamers is a calorimetric method that analyzes UV absorbance at 320 and 620 nm resulting from complexation of poloxamer with cobalt (II) thiocyanate.

  Yun Mao et al. (Journal of Pharmaceutical and Biomedical Analysis, 35 (2004), 1127) described poloxamers using size exclusion chromatography (SEC) and refractive index (RI) detection using THF as a mobile phase column. Describes the measurement method. This method has been applied to the pharmaceutical formulations Avapro, Neurontin and Sudafed, but such active ingredients are “small molecules”. Small molecules are conveniently separated from high molecular weight poloxamers by SEC.

  Size exclusion chromatography (SEC), also known as gel permeation chromatography (GPC), uses porous particles to separate molecules of different sizes. This method is commonly used to separate polymer molecules and to measure the molecular weight and molecular weight distribution of the polymer. Since molecules smaller than the pore size can enter the particle, the path becomes longer and the transit time is longer than a large molecule that cannot enter the particle. All molecules larger than the pore size will elute together without being retained. The average residence time within a particle of molecules that can enter the pores depends on the size and shape of the molecule. Therefore, for different molecules, the total transit time of the column will also be different.

  No method has yet been provided that allows the quantification of poloxamers in protein samples having a molecular weight comparable to poloxamers.

  In particular, for the case where the protein has a molecular weight of 5 to 70 kDa, preferably 20 to 70 kDa, there is a need for a method for quantifying poloxamers in protein samples.

  The present invention relates to a method allowing the quantification of poloxamers in protein samples, in particular liquid protein samples such as liquid pharmaceutical formulations. In particular, the present invention provides a method for quantifying poloxamers in protein samples. That is, the amount of poloxamer in the preparation can be measured at any time during the shelf life of about 2 years of the protein preparation.

The method for quantifying poloxamer in a liquid protein sample comprises:
(A) separating components in a liquid protein sample using a size exclusion chromatography column; and (b) detecting a poloxamer by analyzing the refractive index;
Comprising the steps of:

Preferably, the present invention is a method for quantifying poloxamer in a liquid protein sample comprising:
(A) a separation step using a size exclusion chromatography column;
(B) an elution step using a mobile phase; and
(C) optional poloxamer detection step;
The present invention relates to a method comprising the step of:

  That is, poloxamers can be separated from other components by combining size exclusion chromatography with an elution step using a mobile phase.

  The detection of poloxamers is carried out in a further step, for example by analyzing the elution phase using an RI (refractive index) detection system.

Abbreviations The following abbreviations are used in the description of the present invention.
FSH: follicle stimulating hormone;
r-FSH; r-LH; r-hCG; r-GH; r-IFN-β, r-TSH: recombinant FSH, LH, hCG, GH, INF-β, TSH;
hFSH: human FSH;
r-hFSH: Recombinant human FSH
RI: refractive index KD or Kd or kDa: kilodalton SEC: size exclusion chromatography RT: room temperature WFI: distilled water poloxamer 188 for injection (Poloxamer 188): Another name for Pluronic F68 (BAlu).

  The present invention relates to a simple method that allows the quantification of poloxamers (which are surfactants) in protein samples. The protein sample is preferably a liquid protein sample. The liquid protein sample may be in any form of liquid preparation, but is preferably a liquid pharmaceutical preparation described below. According to one embodiment, liquid pharmaceutical protein samples are contained in single or multiple dose vials.

  According to a further embodiment, the protein sample to be analyzed is lyophilized and dissolved in a suitable aqueous solvent prior to analysis.

The method for quantifying poloxamer in a liquid protein sample comprises:
(A) separating components in a liquid protein sample using a size exclusion chromatography column; and (b) detecting a poloxamer by analyzing the refractive index;
Comprising the steps of:

Preferably, the present invention is a method for quantifying poloxamer in a liquid protein sample comprising:
(A) a separation step using a size exclusion chromatography column;
(B) an elution step using a mobile phase; and
(C) optional poloxamer detection step;
A method comprising the steps of:

  Typically, the size exclusion column will be a SE-HPLC column.

  According to one embodiment, the poloxamer is Poloxamer 188.

  According to a preferred embodiment, the molecular mass of the protein in the liquid protein sample is comparable to the mass of the poloxamer.

  According to a preferred embodiment, the protein in the liquid protein sample has a molecular mass of 5-70 kDa, more preferably 20-70 kDa.

  The ratio of the protein mass to the mass of each poloxamer is preferably 1: 3 to 10: 1, preferably 1: 2 to 1: 7.

  Examples of proteins according to the invention include mammalian proteins such as growth hormones such as human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoprotein; α-1-antitrypsin Insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; chorionic gonadotropin; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrand factor; Anticoagulant factor; atrial natriuretic factor; pulmonary surfactant; plasminogen activator such as urokinase or tissue plasminogen activator (t-PA); bombagene; thrombin; tumor necrosis factor-α and -β; Human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Muller inhibitor; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin (regulated on activation normally T-cell expressed and secreted) Related proteins; DNase; Inhibin; Activin; Vascular endothelial growth factor (VEGF); Hormone or growth factor receptor; Integrin; Protein A or D; Rheumatoid factor; Bone-derived neurotrophic factor (BDNF); Neurotrophic factors such as 4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors such as NGF-P; platelet derived growth factor (PDGF); fibroblast growth factors such as aFGF and bFGF, especially FGF-18; epidermal growth factor ( GF); transforming growth factor (TGF) such as TGF-α and TGF-β including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor I and − II (IGF-I and IGF-II); des (1-3) -IGF-1 (des (1-3) -IGF-1) (brain IGF-I); insulin-like growth factor binding protein; CD3, CD4 CD proteins such as CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO); osteoinductor; osteopontin; immunotoxin; bone morphogenetic protein (BMP); interferons such as interferon-α, -β and -γ Colony stimulating factors (CSF) such as M-CSF, GM-CSF, and G-CSF; interleukins such as IL-1 to IL-10 (IL Superoxide dismutase; T cell receptor; surface membrane protein; decay accelerating factor (DAF); viral antigen such as part of AIDS envelope; transport protein; homing receptor; addressin; regulatory protein; immunoadhesin; In addition, biologically active fragments or variants of any of the polypeptides listed above can be mentioned.

  Among them, proteins according to the present invention include follicle stimulating hormone (FSH), chorionic gonadotropin (CG), luteinizing hormone (LH), interferon-β (IFN-β), PEGylated interferon-β (IFN-β), Preferably it is selected from growth hormone (GH), thyroid stimulating hormone (TSH), fibroblast growth factor 18 (FGF-18) or osteopontin.

  FSH, CG, LH and TSH are glycoproteins classified into gonadotropins. Gonadotropins are used to treat infertility.

  INF-β is a glycoprotein classified as an interleukin. IFN-β is used for the treatment of multiple sclerosis.

  PEG-INF-β is INF-β derived from a polyethylene glycol chain. This improves stability.

  Growth hormone is a non-glycosylated protein. Used in the treatment of growth hormone deficiency in children or adults.

  FGF-18 is used to treat osteoarthritis.

  Osteopontin is a glycosylated single chain polypeptide.

  According to a preferred embodiment, the protein is a heterodimer such as gonadotropin (FSH, LH, CG, TSH and variants). According to a further embodiment, the protein is growth hormone (GH) or interferon-β (IFN-β, or a variant such as a PEGylated variant). According to another further embodiment, said protein is FGF-18 or osteopontin.

  According to a preferred embodiment, the liquid protein sample contains one or more therapeutic proteins. Such samples preferably do not contain non-protein therapeutic agents such as low molecular weight compounds.

  As used herein, follicle stimulating hormone (or FSH) refers to FSH made as a full-length mature protein. An example includes, but is not limited to, human FSH (ie, “hFSH”). It does not matter whether it is produced by recombination or isolated from human-derived materials (eg, postmenopausal female urine).

  This method is applicable not only to natural proteins but also to recombinant proteins. According to one embodiment, the protein formulation is human recombinant FSH, LH, CG, TSH, GH or IFN-β.

  Follicle-stimulating hormone (FSH) is a glycoprotein classified as gonadotropins. FSH is used for treatment of infertility and reproductive disorders (eg, induction of sperm production in men with sperm reduction) for both female and male patients.

  Luteinizing hormone (LH) is a gonadotropin secreted from the anterior pituitary gland. LH is used in female patients in combination with FSH for OI (ovulation induction) and COH (superovulation induction), especially for patients with very low endogenous LH levels, patients with resistance to LH, such as It is used for women suffering from pituitary dysfunction reproductive dysfunction (HH, WHO group I), elderly patients (ie, 35 years of age and older), and patients who have problems with implantation or early miscarriage.

  Chorionic gonadotropin (CG) is gonadotropin produced from the placenta and is obtained from the urine of pregnant women. CG acts on the same receptor as LH and induces the same response. CG is usually used as a long-term source of LH activity because it has a longer circulating half-life than LH. Using CG for OI and COH therapy mimics the natural LH peak and induces ovulation. At the end of stimulation with FSH or with a mixture of FSH and LH, ovulation is induced using injections of human chorionic gonadotropin (hCG). In addition, in the stimulation period for OI and COH, LH activity may be imparted during stimulation of a patient who desires LH activity as described above by using CG in combination with FSH.

  FSH, LH and CG are members of the heterodimeric glycoprotein hormone family. Such families also include thyroid stimulating hormone (TSH). Members of this family are heterodimers and comprise an α-subunit and a β-subunit. These subunits are supported by each other by non-covalent interactions. Human FSH (hFSH) heterodimers are (i) other human family members (ie, chorionic gonadotropin (“CG”), luteinizing hormone (“LH”), and thyroid stimulating hormone (“TSH”)). ) Common glycoprotein α-subunit of 92 amino acids; and (ii) 111 β-amino acid mature β-subunit unique to FSH. The human LH heterodimer consists of (i) the α-subunit of a mature glycoprotein consisting of 92 amino acids as described above; and (ii) a mature β-subunit consisting of 112 amino acids that is unique to LH. Consists of units. Glycoprotein α-subunits and β-subunits tend to be degraded during formulation by interaction with preservatives, surfactants and other excipients. Degradation of subunits results in a decrease in biological efficacy.

  FSH is formulated for intramuscular (IM) or subcutaneous (SC) injection. According to one embodiment, FSH is supplied in lyophilized (solid) form in vials or ampoules at 75 IU / vial and 150 IU / vial and has a shelf life of one and a half years to two years when stored at 2-25 ° C. Have The solution for injection is prepared by re-dissolving the lyophilized product in distilled water for injection (WFI). In addition, liquid FSH formulations comprising 22 μg / 0.5 ml, 33 μg / 075 ml or 66 μg / 1.5 ml FSH and poloxamer 188, sucrose, buffer, methionine and m-cresol are available (Gonal -F pen).

  Thus, FSH has been formulated as a solution in both single and multiple dose forms in vials or ampoules. Single dosage forms must maintain stability and efficacy upon storage prior to use. The multiple dose format not only maintains stability and efficacy during storage prior to use, but also maintains stability and efficacy throughout the entire dose period of the multiple dose regimen after opening the ampoule and It must be maintained in a relatively small state. For this reason, multiple dosage forms usually contain bacteriostatic agents such as benzyl alcohol and m-cresol.

LH is formulated for intramuscular (IM) or subcutaneous (SC) injection. According to one embodiment, LH is supplied in lyophilized (solid) form at 75 IU / vial in a vial or ampoule and has a shelf life of 1 year and a half to 2 years when stored at 2-25 ° C. The solution for injection is prepared by re-dissolving the lyophilized product in distilled water for injection (WFI). Luveris ^™ includes sucrose, buffer, polysorbate 20, and methionine as excipients in addition to LH. Recently, an LH preparation containing poloxamer 188 has been reported (International Publication WO 2004/087213).

Liquid pharmaceutical compositions containing hCG are also commercially available. For example, Ovitrelle ^(trademark) containing mannitol, methionine, poloxamer 188 in a phosphate buffer at pH 7 can be mentioned.

  The expression “variant” is different from human FSH, LH, CG, TSH, IFN-β or GH in that the amino acid sequence, glycosylation pattern, or binding between subunits is different, but FSH, LH, CG, TSH, It is intended to include molecules that exhibit biological activity corresponding to IFN-β or GH. An example is CTP-FSH. This is a long-acting modified recombinant FSH consisting of a wild-type α-subunit and a hybrid β-subunit in which a carboxy terminal peptide of hCG is fused to the C-terminus of the β-subunit of FSH. LaPolt et al .; Endocrinology; 1992, 131, 2514-2520, or Klein et al .; Development and characterization of a long-acting r-hFSH agonist; Human Reprod. 2003, 18, 50-56, etc. Has been. Moreover, single chain CTP-FSH is also mentioned. This is a single-stranded molecule consisting of the following sequence (from N-terminus to C-terminus).

  Here, as described in Klein et al. (Pharmacokinetics and pharmacodynamics of single-chain recombinant human follicle-stimulating hormone containing the human chorionic gonadotrophin carboxyterminal peptide in the rhesus monkey; Fertility &Sterility; 2002, 77, 1248-1255), βFSH is , Represents the β-subunit of FSH, βhCG CTP (113-145) represents the carboxy-terminal peptide of hCG, and αFSH represents the α-subunit of FSH. Other examples of FSH variants include α-subunits and / or β--disclosed in WO 01/58493 (Maxygen) (particularly disclosed in claims 10 and 11 of WO 01/58493). Examples include FSH molecules in which a further glycosylation site is incorporated in the subunits, and FSH molecules having an S—S bond between subunits as described in International Publication WO98 / 58957.

  The FSH variants referred to herein also include a carboxy-terminal deletion of the β-subunit that is shorter than the full-length mature FSH protein. The FSH heterodimer or FSH variant heterodimer can be produced by any suitable method. Such methods include recombinant techniques, (where appropriate) isolation or extraction from natural materials, chemical synthesis, or any combination thereof.

  “Variants” also include PEGylated forms of proteins.

  The term “recombinant” is used to describe the preparation of FSH, LH, CG, TSH, GH, IFN-β or variants made through the use of recombinant DNA technology (eg, International Publication No. WO85 / 01958). See). As an example of a method for expressing FSH or LH by a recombinant technique, a DNA sequence encoding the α- and β-subunits of FSH or LH as described in European Patents EP 0 211 894 and EP 0 487 512 is used. And a method of transfecting a eukaryotic cell with a single vector or with two vectors (providing each subunit individually with a promoter). Another example of producing FSH or LH by recombinant technology is the endogenous sequence encoding the subunit of FSH or LH, as described in European Patent EP 0 505 500 (Applied Research Systems ARS Holding NV). And a method of inserting heterologous regulatory segments by homologous recombination so that they are operatively linked.

  The FSH or FSH variant used according to the present invention can be extracted not only by recombinant means (eg, from mammalian cells etc.) but also from other biological sources (eg urine etc.). Preferred methods include Hakola, K. Molecular and Cellular Endocrinology, 127: 59-69, 1997, Keene et al., J. Biol. Chem., 264: 4769-4775, 1989, Cerpa-Poljak et al., Endocrinology. , 132: 351-356, 1993, Dias et al., J. Biol. Chem., 269: 25289-25294, 1994, Flack et al., J. Biol. Chem., 269: 14015-14020, 1994, Valove et al., Endocrinology, 135: 2657-2661, 1994, US Pat. No. 3,119,740, and US Pat. No. 5,767,067.

  Luteinizing hormone (or LH) as used herein refers to FSH made as a full-length mature protein. An example is human LH, but is not limited thereto. It does not matter whether it is produced by recombination or isolated from human-derived material (eg postmenopausal female urine). The protein sequence of the α-subunit of human glycoprotein is shown in SEQ ID NO: 1, and the protein sequence of the human LH β-subunit is shown in SEQ ID NO: 6. According to a preferred embodiment, LH is recombinant.

  The expression “LH variant” is intended to encompass molecules that differ in amino acid sequence, glycosylation pattern, or binding between subunits from human LH but exhibit LH activity.

  The LH heterodimer or LH mutant heterodimer can be produced by any suitable method. Such methods include recombinant techniques, (where appropriate) isolation or extraction from natural materials, chemical synthesis, or any combination thereof.

  The liquid protein sample of the present invention also includes a mixture of FSH / LH and a variant (International Publication No. WO 2004/087213), and further FSH, hCG, and a variant (International Publication No. WO 2004/105788). ) Is also included.

  The term “aqueous diluent” refers to a liquid solvent containing water. The aqueous solvent system may consist of water alone or may consist of water and one or more water-miscible solvents. Also, solutes such as sugars, buffers, salts or other excipients may be dissolved. More commonly used non-aqueous solvents are short chain organic alcohols such as methanol, ethanol and propanol, short chain ketones such as acetone, and polyhydric alcohols such as glycerol.

  The term “bacteriostatic” or “bacteriostatic agent” refers to a compound or composition that is added to a formulation and acts as an antibacterial agent. Preservative preparations according to the present invention containing FSH or FSH variants or FSH and LH are in accordance with statutory or regulatory guidelines for antiseptic effects as commercially viable multi-use products (preferably for humans) It is preferable to match. Examples of bacteriostatic agents include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, dehydro Examples include sodium acetate and thimerosal.

  The terms “buffering agent” or “physiologically acceptable buffering agent” are known to be safe during formulation in pharmaceutical or veterinary applications and fall within the pH range desired for the formulation. Refers to a solution of a compound having the effect of maintaining or regulating Acceptable buffers that adjust pH from moderately acidic pH to moderately basic pH include, but are not limited to, compounds such as phosphoric acid, acetic acid, citric acid, arginine, TRIS, and histidine. It is not a thing. “TRIS” refers to 2-amino-2-hydroxymethyl-1,3, -propanediol, and any pharmacologically acceptable salt thereof. A preferred buffer is a phosphate buffer using saline or an acceptable salt.

  The term “phosphate buffer” refers to a solution containing phosphoric acid or a salt thereof and adjusted to the desired pH. Phosphate buffers are generally prepared from phosphate or phosphate. Phosphate includes but is not limited to sodium and potassium salts. Several types of phosphates are known in the art, including sodium and potassium salts of primary, secondary and tertiary phosphates. Phosphate is also known to occur as a hydrate of existing salts. The phosphate buffer may cover a range of pH. This range is, for example, from about pH 4 to about pH 10, but the preferred range is from about pH 5 to about pH 9, the most preferred range is from about 6.0 to about 8.0, and most preferably about pH 7.0.

  “Vial” is a term that broadly refers to a container suitable for holding FSH in a solid or liquid state in a sterile state. Examples of vials used herein include FSH to patients via ampoules, cartridges, blister packages, or syringes, pumps (including osmotic pumps), catheters, transdermal patches, lungs or transmucosal sprays. Other containers suitable for delivery include. Vials suitable for packaging products for parenteral, pulmonary, transmucosal, or transdermal administration are well known and recognized in the art.

  The expression “multi-dose use” means that a single vial, ampoule or cartridge of an FSH formulation or protein formulation can be injected into multiple injections, eg 2, 3, 4, 5, 6 or more times. Including the use of injections. Injection is preferably performed over a period of at least about 12 hours, such as about 24 hours, and preferably over a period of up to about 12 days. For example, a time interval such as 6, 12, 24, 48, or 72 hours may be provided as a time interval between injections.

  The term “stability” refers to the physical, chemical, and conformational stability of a given protein in the formulations of the present invention (including maintaining biological efficacy). Causes of protein formulation destabilization include formation of higher order polymers by chemical degradation or aggregation of protein molecules, degradation of heterodimers into monomers, deglycosylation, glycosylation modification, oxidation (especially α- Oxidation of subunits in heterodimers) or any other structural modification that reduces at least one biological activity of a polypeptide included in the invention.

  A “stable” solution or formulation is such that the degree of degradation, modification, aggregation, loss of biological activity, etc. of the protein contained therein is adjusted to an acceptable level and is not acceptable. It is a solution or formulation that does not increase over time. The formulation preferably maintains at least about 80% of the indicated protein activity for a period of up to 2 years.

  The object underlying the present invention is to provide a simple and rapid method for measuring the amount of poloxamer in a protein sample. Poloxamers can be used as surfactants and are selected from block copolymers of ethylene oxide and propylene oxide. Preferred are Pluronic® F77, Pluronic F87, Pluronic F88 and Pluronic® F68, particularly preferably Pluronic F68 (BASF, Pluronic F68, also known as Poloxamer 188). Is).

  As mentioned above, pharmaceutical formulations are required to have a shelf life of up to 2 years at a storage temperature of 2-25 ° C. This suggests the likelihood that the formulation will remain stable over the period. Since various excipients contained in liquid formulations directly affect the stability of protein formulations or indirectly through degradation, analytical tools to evaluate the stability of the formulations are required.

  More specifically, the poloxamer surfactant is a block copolymer of ethylene oxide (EO) and propylene oxide (PO). The propylene oxide block (PO) is sandwiched between two ethylene oxide (EO) blocks.

Poloxamer synthesis is:
Creating a hydrophobic substance of the desired molecular weight by the controlled addition of propylene oxide to the two hydroxyl groups of propylene glycol;
-It is carried out in a two-stage process in which ethylene oxide is added and the hydrophobic substance is sandwiched between hydrophilic groups.

  Poloxamer surfactants are also known as pluronics.

  In Pluronic (registered trademark) F77, the ratio of polyoxyethylene (hydrophilic substance) is 70%, and the molecular weight of hydrophobic substance (polyoxypropylene) is about 2,306 Da.

  In Pluronic F87, the ratio of polyoxyethylene (hydrophilic substance) is 70%, and the molecular weight of hydrophobic substance (polyoxypropylene) is about 2,644 Da.

  In Pluronic F88, the ratio of polyoxyethylene (hydrophilic substance) is 80%, and the molecular weight of hydrophobic substance (polyoxypropylene) is about 2,644 Da.

  In Pluronic F68, the proportion of polyoxyethylene (hydrophilic substance) is 80%, and the molecular weight of hydrophobic substance (polyoxypropylene) is about 1,967 Da.

Typical characteristics of Pluronic F77 are listed below:
Average molecular weight: 6600;
Melting point / pour point: 48 ° C .;
Physical form at 20 ° C .: Solid viscosity (Brookfield) cps: 480 [liquid at 25 ° C., paste at 60 ° C., solid at 77 ° C.];
Surface tension, dyne / cm at 25 ° C .;
Concentration 0.1%: 47.0
Concentration 0.01%: 49.3
Concentration 0.001%: 52.8
Interfacial tension, dyne / cm at 25 ° C. vs. Nujol;
Concentration 0.1%: 17.7
Concentration 0.01%: 20.8
Concentration 0.01%: 25.5
Draves Wetting, sec 25 ° C
Concentration 1.0%:> 360
Concentration 0.1%:> 360
Foam height Ross Miles, 0.1%, mm at 50 ° C: 100
Ross Miles, 0.1%, mm at 26 ° C: 47
Dynamic, 0.1%, mm at 400 ml / min:> 600
Cloud point in aqueous solution, ° C
Concentration 1%:> 100
Concentration 10%:> 100
HLB (hydrophilic-oil balance): 25

The typical characteristics of Pluronic F87 are listed below:
Average molecular weight: 7700;
Melting point / pour point: 49 ° C .;
Physical form at 20 ° C .: Solid viscosity (Brookfield) cps: 700 [liquid at 25 ° C., paste at 60 ° C., solid at 77 ° C.];
Surface tension, dyne / cm at 25 ° C .;
Concentration 0.1%: 44.0
Concentration 0.01%: 47.0
Concentration 0.001%: 50.2
Interfacial tension, dyne / cm at 25 ° C. vs. Nujol;
Concentration 0.1%: 17.4
Concentration 0.01%: 20.3
Concentration 0.01%: 23.3
Draves Wetting, sec 25 ° C
Concentration 1.0%:> 360
Concentration 0.1%:> 360
Foam height Ross Miles, 0.1%, mm at 50 ° C: 80
Ross Miles, 0.1%, mm at 26 ° C: 37
Dynamic, 0.1%, mm at 400 ml / min:> 600
Cloud point in aqueous solution, ° C
Concentration 1%:> 100
Concentration 10%:> 100
HLB (hydrophilic-oil balance): 24

The typical characteristics of Pluronic F88 are listed below:
Average molecular weight: 11400;
Melting point / pour point: 54 ° C .;
Physical form at 20 ° C .: Solid viscosity (Brookfield) cps: 2300 [liquid at 25 ° C., paste at 60 ° C., solid at 77 ° C.];
Surface tension, dyne / cm at 25 ° C .;
Concentration 0.1%: 48.5
Concentration 0.01%: 52.6
Concentration 0.001%: 55.7
Interfacial tension, dyne / cm at 25 ° C. vs. Nujol;
Concentration 0.1%: 20.5
Concentration 0.01%: 23.3
Concentration 0.01%: 27.0
Draves Wetting, sec 25 ° C
Concentration 1.0%:> 360
Concentration 0.1%:> 360
Foam height Ross Miles, 0.1%, mm at 50 ° C: 80
Ross Miles, 0.1%, mm at 26 ° C: 37
Dynamic, 0.1%, mm at 400 ml / min:> 600
Cloud point in aqueous solution, ° C
Concentration 1%:> 100
Concentration 10%:> 100
HLB (hydrophilic-oil balance): 28

The typical characteristics of Pluronic F68 are listed below:
Average molecular weight: 8400;
Melting point / pour point: 52 ° C .;
Physical form at 20 ° C .: Solid viscosity (Brookfield) cps: 1000 [liquid at 25 ° C., paste at 60 ° C., solid at 77 ° C.];
Surface tension, dyne / cm at 25 ° C .;
Concentration 0.1%: 50.3
Concentration 0.01%: 51.2
Concentration 0.001%: 53.6
Interfacial tension, dyne / cm at 25 ° C. vs. Nujol;
Concentration 0.1%: 19.8
Concentration 0.01%: 24.0
Concentration 0.01%: 26.0
Draves Wetting, sec 25 ° C
Concentration 1.0%:> 360
Concentration 0.1%:> 360
Foam height Ross Miles, 0.1%, mm at 50 ° C .: 35
Ross Miles, 0.1%, mm at 26 ° C: 40
Dynamic, 0.1%, mm at 400 ml / min:> 600
Cloud point in aqueous solution, ° C
Concentration 1%:> 100
Concentration 10%:> 100
HLB (hydrophilic-oil balance): 29

  Other poloxamer polymers with similar properties to the poloxamers listed above can also be used in the formulations of the present invention. A preferred poloxamer surfactant present in the protein preparation to be analyzed by this method is Pluronic F68 (= Poloxamer 188).

  The concentration of pluronic in the liquid protein formulation, in particular the concentration of pluronic F68, is preferably from about 0.01 mg / ml to about 1 mg / ml, more preferably from about 0.05 mg / ml to about 0.5 mg / ml. Particularly preferred is about 0.2 mg / ml to about 0.4 mg / ml, most preferred about 0.1 mg / ml.

  The pH of the protein formulation analyzed according to the method of the present invention is about 6.0 to about 8.0, more preferably about 6.8 to about 7.8, such as about pH 7.0, about pH 7.2, and about 7.4. Preferred buffering agents include phosphoric acid, and preferred counter ions include sodium ions or potassium ions. Phosphate saline buffer is well known in the art and includes Dulbecco's phosphate buffered saline and the like. The buffer concentration in the total solution can be selected between about 5 Mm, about 9.5 Mm, about 10 Mm, about 50 Mm, about 100 Mm, about 150 Mm, about 200 Mm, about 250 Mm, and about 500 Mm. Among them, a sound having a buffer concentration of about 10 Mm is preferable. A buffer of phosphate ion 10Mm, pH 7.0 is particularly preferred.

  The pH of the FSH formulation analyzed according to the method of the present invention is preferably about 6.0 to about 8.0, more preferably about 6.8 to about 7.8, such as about pH 7.0, about pH 7.2, And about 7.4. Preferred buffering agents include phosphoric acid, and preferred counter ions include sodium ions or potassium ions.

  The pH of the combined formulation of FSH and LH analyzed according to the method of the present invention is about 6.0 to about 9.0, more preferably about 6.8 to about 8.5, such as about pH 7.0, about pH 8. 0, and about 8.2, most preferably about pH 8.0.

  The pH of the hCG formulation analyzed according to the method of the present invention is preferably about 6.0 to about 8.0, more preferably about 6.8 to about 7.8, such as about pH 7.0, about pH 7. 2 and about 7.4.

  The protein sample is preferably a single-dose or multi-dose liquid formulation. Liquid formulations intended for use in multiple doses according to the present invention include phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc. ), Thymol, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal. Particularly preferred are phenol, benzyl alcohol and m-cresol, more preferred are phenol and m-cresol, and most preferred is m-cresol. These bacteriostatic agents are used in amounts that produce a concentration effective to maintain the formulation substantially sterile (appropriate for injection) over the period of multiple dose injection. Such a period is, for example, about 12 hours or about 24 hours to about 12 days or about 14 days, preferably about 6 days to about 12 days. The amount of bacteriostatic agent present is preferably about 0.1% (amount of bacteriostatic agent / amount of solvent) to about 2.0%, more preferably about 0.2% to about 1.0%. To do. In the case of benzyl alcohol, a particularly preferred concentration is 0.9%. In the case of phenol, a particularly preferred concentration is about 0.5%. In the case of m-cresol, a particularly preferred concentration is about 0.3% (eg, about 3 mg / ml in WFI).

  Size exclusion columns are well known to those skilled in the art and are selected for each protein and poloxamer in the sample. A polymer matrix should be used as the column gel. Preferred columns include SE-HPLC columns from Toso Haas, trade name TSK G3000PW. The beads of this matrix have a particle size of 10 or 17 μm and a pore diameter of about 200 mm. This column is commercially available.

  The detection step can be performed by any RI detection system known to those skilled in the art.

  It has been found that separation of poloxamers from proteins becomes easier when the conditions are made more acidic.

  Thus, the mobile phase used in the method according to the invention is an aqueous solvent such as water or a buffer solution.

  According to certain embodiments, the pH of the mobile phase is adjusted to less than 7, preferably less than 3, more preferably between about 1.6 and 2.0, most preferably between 1.9 and 2.0. The According to one embodiment, the protein is a heterodimer such as FSH, CG, LH or TSH. Under acidic conditions, the heterodimeric protein is broken down into subunits and facilitates migration, improving the separation and detection steps (resolution). Those skilled in the art can select an acidic agent suitable for adjusting the pH. The most preferred acid is trifluoroacetic acid (TFA).

  In general, a sample to be evaluated is prepared and injected into a column. By taking into account the refractive index in the detection step of the method, the resulting chromatogram contains at least one additional peak for proteins and excipients in addition to the poloxamer peak area. By evaluating the peak area, the poloxamer present in the analytical sample can be quantified. A calibration curve of poloxamer necessary for quantification has been prepared (see Examples).

  Subsequently, the present invention is illustrated by examples.

Example 1 (see FIG. 1)
The purpose of this example is to store a sample of a commercially available liquid hCG formulation, Ovitrelle ^™ , at room temperature for 18 months and then analyze the poloxamer 188 concentration in the sample. That is, the stability of the liquid formulation with respect to poloxamer 188 was evaluated. The concentration of poloxamer 188 at the time of preparation was about 100 μg / ml. The quantification protocol for poloxamer 188 in Ovitrelle ^™ was as follows.

The ingredients of the injectable liquid Ovitrelle ^™ were chorionic gonadotropin-α, mannitol, L-methionine, poloxamer 188, phosphoric acid, NaOH and water.

2. Method 2.1 Eluent A (H ₂ O / TFA 0.5%)
To 950 ml of purified water in a 1 liter graduated cylinder, 5 mL of trifluoroacetic acid (TFA) was added and adjusted to 1000 ml with stirring. The pH of the eluent was 1.7 to 1.9.

2.2 Eluent B (20% ethanol)
To 750 ml of purified water in a 1 liter graduated cylinder, 200 ml of ethanol was added and adjusted to 1000 ml with stirring.

2.3 Cleaning solution for autosampler (10% methanol)
To 850 ml of purified water in a 1 liter graduated cylinder, 100 ml of methanol was added and adjusted to 1000 ml with stirring.

2.4 Seal washing solvent (5% methanol)
50 ml of methanol was added to 900 ml of purified water in a 1 liter graduated cylinder and adjusted to 1000 ml with stirring.

2.5 Dilution solution for calibration curve (liquid formulation without poloxamer 188)
To 850 ml of purified water in a 1 liter graduated cylinder was added 54.6 g D (-) mannitol, 0.98 g ortho-phosphate 85%, 200 μg L-methionine. 50% sodium hydroxide was added dropwise to adjust the solution to pH 7.0 and the volume was adjusted to 1 ml. This solution was filtered through a 0.45 μm filter.

  Instead of the above, water may be used as a diluted solution for the calibration curve.

2.6 Concentrated solution for calibration curve 200 mg of poloxamer 188 was dissolved in 80 ml of purified water in a 100 ml graduated cylinder and the volume was adjusted to 100 ml. A calibration curve was created based on the expected concentration in the sample to be analyzed. In this example, the predicted poloxamer 188 concentration in Ovitrelle ^{™ of the} liquid for injection was about 100 μg / ml, so the calibration curve was in the range of 50 to 160 μg / ml.

3 Sample Preparation Blank 0.05 ml of calibration curve dilution solution was injected.

Samples All samples were subjected to testing without any pretreatment and 0.05 ml was injected.

4). Operating conditions 4.1. Instrument settings The following solutions were connected to HPLC tubes.
Tube A: Eluent A (H ₂ O / TFA 0.5%)
Tube B: Eluent B (20% ethanol)

  The apparatus was purged by filling tubes A and B and rinsed for 3 minutes at a flow rate of 2 mL / min. If there was a deaerator on the tube, it was turned on. For tube A, the solenoid valve of the flowing RI detector was switched to purge mode for at least 30 minutes prior to analysis. The flow cell was purged and a fresh mobile phase was poured into the reference side of the cell.

4.2. Column equilibration Eluent A was allowed to flow through the column to equilibrate the column. Equilibration was complete when the baseline was stable.

4.3. Storage column After the analysis was completed, the column was rinsed with at least 30 ml of purified water and then with 30 ml of 20% ethanol.

4.4. Measurement of Poloxamer 188 Concentration in Obitrel Sample The linear regression method was used as a model to calculate the poloxamer 188 concentration in the Obitrel sample.

Y = a + bx
here,
Y = total area of poloxamer 188 a = intercept b = slope x = poloxamer 188 concentration in μg per ml injected.

The sample was integrated as shown in FIG.
The intercept (a) and slope (b) of the calibration curve were calculated, and linear regression was calculated with software called Statgraphics plus.
The concentration of poloxamer 188 was calculated applying the following formula:

  The solution of Equation 1 represents the poloxamer 188 concentration in each obitrell sample. The unit is μg / ml.

  The poloxamer concentration (see FIG. 1) in the sample of this example was about 94 μg / ml.

Example 2
The purpose of this example is to store a sample of a commercial liquid hFSH formulation, Gonal-F RFF Pen ^™ , at room temperature for 18 months and then analyze the poloxamer 188 concentration in the sample. That is, the stability of the liquid formulation with respect to poloxamer 188 was evaluated. The poloxamer 188 concentration at the time of preparation was about 100 μg / ml. The protocol for quantifying poloxamer 188 in Gonal-F RFF Pen ^™ was identical to the protocol shown for Obitrel in Example 1 except that the column flow rate was 0.75 ml / min.
Poloxamers were successfully separated from hFSH and quantified individually.

Example 3
The purpose of this example is to analyze the poloxamer 188 concentration in a sample of a commercially available liquid hGH formulation, Serostim ^™ . The protocol was the same as in Example 2.
Poloxamers were successfully separated from hGH and quantified individually.

Example 4
The purpose of this example is to analyze the poloxamer 188 concentration in a sample of a commercially available liquid hIFN-β formulation. The protocol was the same as in Example 2.
Poloxamers were successfully separated from hIFN-β and quantified individually.

FIG. 1 shows a chromatogram for the quantification of Poloxamer 188 in hCG formulations. The peak region of poloxamer 188 in this example is present at an elution time of about 14-16 minutes (retention time is a function of flow rate and can vary). Based on the area under the curve, poloxamer 188 in the hCG sample can be quantified.

Claims (14)

A method for quantifying a poloxamer in a liquid sample containing a protein having a molecular weight of 5 to 70 kDa, comprising :
The sample:
(A) a separation step using a size exclusion chromatography column;
(B) an elution step using an acidic pH mobile phase; and
(C) optional poloxamer detection step;
A method comprising the step of:   The method of claim 1, wherein the poloxamer is Poloxamer 188. The method according to claim 1 or 2, wherein the protein has a molecular weight of 20 to 70 kDa.   The method according to claim 1, wherein the protein is a heterodimeric protein.   5. The method of claim 4, wherein the protein is a gonadotropin selected from FSH, LH, hCG, TSH.   The method according to any one of claims 1 to 3, wherein the protein to be analyzed is interferon-β or growth hormone (GH).   The method according to any one of claims 1 to 6, wherein the sample is an aqueous pharmaceutical composition containing FSH, LH, hCG, TSH, GH or interferon-β. The mobile phase is an aqueous Solvent The method according to any one of claims 1 to 7. 9. The method of claim 8 , wherein the pH of the mobile phase is adjusted to less than 3. The mobile phase has a pH of 1 . The method of claim 9 , wherein the method is adjusted to 9 to 2.0. Detecting step of the poloxamer The method according to any one of encompasses claims 1-10 analyzing the refractive index. The method according to any one of claims 1 to 11 , wherein the size exclusion column is an SE-HPLC column packed with a polymer matrix containing beads. 13. The method of claim 12 , wherein the matrix beads have a particle size of 10 or 17 [mu] m. 14. A method according to claim 12 or 13 , wherein the matrix beads have a pore size of about 200 mm.

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